Method of detecting risk factor for onset of diabetes

ABSTRACT

Risk factors for diabetic onset in a subject are detected by using a gene coding for CD38 protein, which is originally identified as a human lymphocyte surface marker, as a risk gene, and detecting mutations in the gene.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method for the detection of adisease. More particularly, the present invention relates to a methodfor detecting risk factors related to the onset of diabetes in asubject. Description of the Related Art:

[0003] Today, at least 30 million people worldwide are diabetics, andcurrently the number is increasing along with a rapid rise in theelderly population. Thus, there is a firm belief that a further increasein the number of diabetic patients will inevitably occur in the nearfuture. Japan is not exempt from this world trend; at present, about 6million Japanese people are afflicted with diabetes, and in severalyears the number is expected to reach 10 million.

[0004] Diabetes (diabetes mellitus) is a disease that can be describedas follows: Glucose is one of the nutrients important for the body, inparticular for individual cells. However, when glucose is notincorporated into cells effectively, the glucose accumulates in blood,inducing a high blood glucose concentration. As a result, glucose isexcreted into the urine.

[0005] Diabetes can be broadly classified into 3 categories:insulin-dependent diabetes mellitus (IDDM; type I diabetes),non-insulin-dependent diabetes mellitus (NIDDM; type II diabetes), andother types of diabetes called secondary diabetes that are incidentallyinduced by the onset of particular diseases, such as pancreatitis.

[0006] Insulin-dependent diabetes (IDDM; type I diabetes) has its onsetat a relatively young age. After onset, progress is rapid, resulting inaccumulation of metabolites; i.e., ketone bodies, in blood. Withoutcontinuous treatment with insulin, the patient's life is endangered.Thus, this type of diabetes is very serious.

[0007] In contrast, non-insulin dependent diabetes (NIDDM; type IIdiabetes) often has its onset after adulthood is reached. The symptomsgradually progress, starting from abnormalities in intravenous glucosetolerance (IGT). Thus, this type of diabetes progresses relativelyslowly, and treatment with insulin is not always necessary.

[0008] As described above, there are two principal types of diabetes,with two corresponding approaches to treatment. In 1997, the AmericanDiabetes Association (ADA) proposed new classification and diagnosisstandards for diabetes. In Japan also, a new concept of diabetes, newclassification, and standards for diabetes have been discussed. The newclassification is organized on the basis of combinations between thecauses of diabetes and symptoms. The symptoms are by and large the sameas those mentioned above. On the other hand, now, understanding thecauses of diabetes will become more and more important when carrying outdiagnosis and classification. As for the new classification, the mostnoteworthy point is that diabetes caused by genetic abnormalities isincorporated into the third category of “other particular types ofdiabetes.”

[0009] Concerning genes involved in the onset of diabetes, abnormalitiesin the genes encoding insulin, insulin receptors, glucokinase (MODY2),HNF-4α ((MODY1), HNF-1α (MODY3), and mitochondria have so far beenknown. However, the incidence rates of such abnormalities have been lowin all cases. For example, although the mutation from adenosine toguanine at nucleotide position 3243 of the mitochondrial DNA is found ata relatively high incidence rate as compared with other genes involvinggene abnormalities, the corresponding mutation is found in less than 1%of the NIDMM cases; and even when the other mutations in themitochondria are all added together, the total mutation incidence rateis considered to reach a level of about 2%. Thus, it may be concludedthat the above-mentioned gene mutations can explain only why a tinyproportion of a large number of diabetic patients contract thecondition. In addition, it is generally regarded that diabetic onset isinduced by not just a single gene abnormality, but by a multiplicity ofgene abnormalities together with the influence of various environmentalfactors.

[0010] Given the facts cited above, examining and determining thosegenes contributing to the onset of diabetes or risk factors involvingthe onset is important not only in order to carry out diagnosis ofdiabetes but also to facilitate the selection of those individualsbelonging to the high risk group so that counter measures can beintroduced to delay the onset, as well as to find clues which willassist us in understanding the onset mechanisms and thus help us findappropriate therapeutic methods to treat diabetes.

SUMMARY OF THE INVENTION

[0011] Problems to be solved by the present invention are (1) to knowwhat type of diabetes a diabetic individual is presently suffering from;and (2) to find risk genes in order to predict whether or not anindividual who presently appears healthy carries risk factors that arecapable of causing diabetes in the future, and further to predict whattype of diabetes the individual would suffer from in that event (notethat hereafter a factor associated with the above (1) and/or (2) will bealso referred to as a “risk factor for diabetic onset”); and also toprovide means for detecting the risk factors for diabetic onset usingthe risk genes.

[0012] In order to solve the problems, the present inventors havesearched risk genes enabling us to determine risk factors for diabeticonset by analysis thereof, and found that abnormalities of the genecoding for CD38 (hereafter the gene will be also referred to as the CD38gene) induce diabetic onset. CD38 is an enzyme participating in bothproduction and hydrolysis of cyclic ADP ribose (cADPR), a secondmessenger, capable of stimulating insulin secretion upon glucosestimulation in β cells of Langerhans' islands in the pancreas. Thus, thepresent inventors concluded that the gene CD38 is the risk gene enablingus to determine risk factors for diabetic onset; i.e., the gene thepresent inventors have been looking for. The present invention has beenaccomplished on the basis of the above finding.

[0013] Accordingly, the present invention provides a method of detectinggenetic factors (risk factors) in relation to diabetic onset inindividuals through detection of abnormalities in the gene CD38. Themethod may hereafter be referred to as the detection method of thepresent invention.

[0014] The CD38 protein encoded by the gene CD38 is a protein originallyidentified as a human lymphocyte surface marker. The close relationshipbetween the CD38 protein and glucose metabolism in the body has beendiscovered through long-term studies by the present inventors Okamoto,Takasawa, et al.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows an abnormal band pattern obtained from gene mutationanalysis of exon 2 of the CD38 gene by use of the DGGE method.

[0016]FIG. 2 shows an abnormal band pattern obtained from gene mutationanalysis of exon 3 of the CD38 gene by use of the DGGE method.

[0017]FIG. 3 shows an abnormal band pattern obtained from gene mutationanalysis of exon 4 of the CD38 gene by use of the DGGE method.

[0018]FIG. 4 shows an abnormal band pattern obtained from gene mutationanalysis of exon 7 of the CD38 gene by use of the DGGE method.

[0019]FIG. 5 shows an abnormal band pattern obtained from gene mutationanalysis of exon 8 of the CD38 gene by use of the DGGE method.

[0020]FIG. 6 shows nucleotide sequences identified by direct sequencingfor exon 2 of the CD38 gene that exhibited an abnormal band patternunder application of the DGGE method.

[0021]FIG. 7 shows nucleotide sequences identified by direct sequencingfor exon 3 of the CD38 gene that exhibited one abnormal band patternunder application of the DGGE method.

[0022]FIG. 8 shows nucleotide sequences identified by direct sequencingfor exon 3 of the CD38 gene that exhibited the other abnormal bandpattern under application of the DGGE method.

[0023]FIG. 9 shows nucleotide sequences identified by direct sequencingfor exon 4 of the CD38 gene that exhibited one abnormal band patternunder application of the DGGE method.

[0024]FIG. 10 shows nucleotide sequences identified by direct sequencingfor exon 4 of the CD38 gene that exhibited the other abnormal bandpattern under application of the DGGE method.

[0025]FIG. 11 shows nucleotide sequences identified by direct sequencingfor exon 7 of the CD38 gene that exhibited an abnormal band patternunder application of the DGGE method.

[0026]FIG. 12 shows nucleotide sequences identified by direct sequencingfor exon 8 of the CD38 gene that exhibited an abnormal band patternunder application of the DGGE method.

[0027]FIG. 13 shows the results of analysis of the exon 3 Arg140Trpmutation by PCR-RFLP.

[0028]FIG. 14 shows the results of analysis of the exon 7 Ser264Leumutation by PCR-RFLP.

[0029]FIG. 15 shows results of analysis of the intron 7 −28G/A mutationby PCR-RFLP.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Before describing a variety of modes of the present invention,below, the relationships between the key player in the presentinvention; i.e., the gene CD38 or the CD38 protein, and glucosemetabolism in the body will first be summarized.

[0031] Glucose is the most important substance for stimulating insulinsecretion from β cells of the Langerhans' islands in the pancreas.Concerning insulin secretion, it is generally regarded that upon theglucose stimulation, the intracellular Ca²⁺ concentration is elevated totrigger insulin secretion. The increase in the intracellular Ca²⁺ levelis accomplished by both the influx of extracellular Ca²⁺ into cells andrecruitment from the intracellular Ca²⁺ pool. Concerning the influx ofextracellular Ca²⁺, the theory of “ATP-sensitive K⁺ channel” proposed byAshcroft et al. is widely accepted. As for the molecular mechanisms, itsdetailed explanations have been provided by Inagaki, Kiyono, et al.(Seikagaku, 69: 1067-1080, 1997).

[0032] In contrast, as for the recruitment of Ca²⁺ from theintracellular Ca²⁺ pool, it has been pointed out that the increase inthe intracellular Ca²⁺ level occurs at the same time or earlier thandoes the influx of extracellular Ca²⁺ from an experiment with humaninsulin-producing cells (Rojas E. et al., Endocrinology, 134: 1771-1781,1994). Further, it has been reported that inositol 1, 4, 5-triphosphate(IP₃) functions as a second messenger for the Ca²⁺ recruitment in manytypes of cells. Similarly, it has been suggested that IP₃ plays animportant role as a second messenger for Ca²⁺ recruitment in theinsulin-producing cells.

[0033] The present inventors Okamoto et al. demonstrated thatmaintaining the intracellular NAD⁺ level in β cells is a prerequisitefor maintaining the insulin-producing function of β cells, showing thatDNA damage is induced by the action of a cytotoxic agent forinsulin-producing β cells, such as streptozotocin, and that, as aresult, the cytotoxic agent causing DNA damage induces activation ofpoly (ADP-ribose) synthetase, which in turn causes intracellular NAD⁺ tobe depleted, resulting in lowering the insulin-producing ability of βcells. In contrast, as they have shown, when lowering of theintracellular NAD⁺ concentration is inhibited by blocking with aninhibitor of poly (ADP-ribose) synthetase, insulin-producing ability ofβ cells is maintained (Yamamoto H, et al., Nature, 294: 284-286, 1981;Okamoto H, et al., 1990, in Molecular Biology of the Islands ofLangerhans, Okamoto H, ed. pp. 209-231, Cambridge University Press,Cambridge; Okamoto H, et al., Biochimie, 77: 356-363, 1995). Based onthis background, concerning the mechanisms of insulin secretioninvolving glucose, the present inventors Okamoto, Takasawa, et al. havesuggested that cADPR synthesized from NAD⁺ is involved in expression ofthe insulin-producing cell function.

[0034] In the case of Ca²⁺ releasing experiments with Fluo3 usingmicrosomes prepared from the Langerhans' islands of rat pancreases,cADPR induces the Ca²⁺ release from the microsomes of Langerhans'islands, and continuous addition of cADPR confirms attenuation of Ca²⁺release. In contrast, upon addition of IP₃, Ca²⁺ release is notobserved. From these results, the present inventors Takasawa, Okamoto,et al. have concluded that the Ca²⁺ release with cADPR from theLangerhans' islands microsomes differs from that with IP₃ (Takasawa S,et al., Science, 259: 370-373, 1993). Further, from radioimmunoassayswith an antibody against cADPR, Takasawa, Okamoto et al. showed that theamount of cADPR in insulin-producing cells is increased upon glucosestimulation (Takasawa S, et al., J. Biol. Chem., 273: 2497-2500, 1998).From these results, Ca²⁺ recruitment upon glucose stimulation observedin the insulin-producing cells is considered to be due mainly to cADPR.

[0035] Further, Takasawa, Okamoto, et al. demonstrated that the CD38protein shows ADP-ribosyl cyclase activity capable of synthesizing cADPRwhen NAD⁺ is used as a substrate, and on the other hand, when cADPR isused as a substrate, the CD38 protein shows cADPR hydrolase activitycapable of synthesizing ADPR (Takasawa S, et al., J. Biol. Chem., 268:26052-26054, 1993). It is well known that the cADPR hydrolysis reactionis suppressed with 2-8 mM ATP in a concentration-dependent manner,whereas, when NAD⁺ is used as a substrate, the amount of cADPRproduction is increased in an ATP concentration-dependent manner.Further, Tohgo, Takasawa et al. demonstrated the molecular mechanism ofsuppression of cADPR hydrolase activity by ATP is due to competitionbetween ATP on the order of mmol units and cADPR for the cADPR bindingsite at residue 129Lys of the CD38 protein (Tohgo A, Takasawa S, et al.,J. Biol. Chem., 272: 3879-3882, 1997). In fact, it has been reportedthat the ATP concentration in the insulin-producing cells is changedwithin a range of 2-8 mM upon glucose stimulation, and further thechange of cADPR amount could be explained by the mechanism that cADPRhydrolysis activity of the CD38 protein is suppressed by ATP, assumingthat the CD38 protein is present in the insulin-producing cells.

[0036] Further, together with Kato and others, Takasawa, Okamoto, et al.have demonstrated that in transgenic mice overexpressing the CD38protein in β cells of the Langerhans' islands, the insulin secretion inresponse to glucose is facilitated significantly as compared with thecontrol mice (J. Biol. Chem. 270, 30045-30050, 1995).

[0037] As described above, the mechanism of insulin secretion isconsidered as follows: cADPR hydrolysis activity of the CD38 protein issuppressed by ATP, which is produced by glucose stimulation, resultingin an increase in the cADPR amount, followed by the Ca²⁺ recruitmentfrom the intracellular Ca²⁺ pool in microsomes, thereby causing insulinsecretion.

[0038] Further, the Ca²⁺ release mechanism by cADPR is considered to bethrough ryanodine receptor (type 2) in endoplasmic reticulum, since theCa²⁺ release with cADPR is cross-desensitized with Ca²⁺ release withryanodine, as discovered by Okamoto, Takasawa, et al. Takasawa, Okamoto,et al. demonstrated that, in the Langerhans' β cells, Ca²⁺/calmodulin(CaM)-dependent protein kinase II phosphorylatesryanodine-type Ca²⁺ -releasing channels (i.e. cADPR-sensitiveCa²⁺-releasing channels) in endoplasmic reticulum. Due to thephosphorylation, the Ca²⁺-releasing sensitivity to cADPR is enhanced toelicit release of Ca²⁺ through the sensitized ryanodine-typeCa²⁺-releasing channels upon glucose stimulation (J. Biol. Chem. 270:30257-30260, 1995). Further, Takasawa, Okamoto, et al. demonstrated thatthe Ca²⁺-releasing with IP₃ dominantly occurs in ob/ob mice, whereas theCa²⁺-releasing with cADPR mainly occurs in normal mice (Takasawa S, etal., J. Biol. Chem., 273: 2497-2500, 1998). Furthermore, it has alsobeen suggested that exhaustion and consumption of intracellular NAD⁺ ininsulin production cause diabetes, since it has been reported thatknock-out mice lacking the poly (ADP-ribose) polymerase (PARP) genegenerated by gene-targeting show resistance to the onset of diabetes tobe induced with streptozotocin (Burkart V, et al., Nature Med., 5:314-319, 1999). In conclusion, Okamoto, Takasawa, et al. demonstratedthat the process involving cADPR production using the NAD⁺ substrate,recruitment of intracellular Ca²⁺, and insulin secretion is importantfor insulin-producing cells, and that the CD38 gene or CD38 protein isintimately involved in the process. Thus, from our results and analyses,the present inventors have come up to the possibility that abnormalitiesof the gene CD38 could be used to detect risk factors for diabeticonset. The gene is closely involved in the onset, however, it should bepointed out that this deduced possibility was very surprising inconsideration that all the genes whose abnormalities were reportedlyassociated with diabetes have not always turned out to be clinicallyuseful as markers for detecting risk factors for diabetic onset.

[0039] The major sites of abnormality of the gene CD38 that can be usedfor the detection method in the present invention are as follows: (1)the site encoding arginine at amino acid residue 140 of the CD38 proteinencoded by the CD38 gene; (2) the site encoding serine at amino acidresidue 264; and (3) guanine at nucleotide position −28 in intron 7.Note that abnormalities at these sites are independent from each otherand are not related. More details of these sites with geneticabnormalities will be described later in the Examples section.

[0040] A variety of modes of the present invention will next bedescribed.

[0041] As described above, the present invention is directed to a methodof detecting risk factors for diabetic onset on the basis of detectionof genetic abnormalities of the gene CD38, a risk gene concerningdiabetic onset.

[0042] The CD38 gene and its encoding CD38 protein have already beenanalyzed [Nata K. et al., Gene, 186: 285-292, 1997; Sequence No. 1(nucleotide and amino acid sequences) and Sequence No. 2 (amino acidsequence)] By examining the correlation between genetic mutations of theCD38 gene and risk factors for diabetic onset, genetic mutations of theCD38 gene useful for the detection method of the present invention canbe found. For this, desirable mutations utilized for the presentinvention can be found by analyzing combinations chosen between“diabetic patients and healthy individuals” and between“insulin-dependent diabetes and non-insulin dependent diabetes”concerning mutation sites of the CD38 gene, their incidences, andfunctions of their encoding proteins. Detailed working protocols will bedescribed hereinbelow in the Examples section.

[0043] As used herein, “genetic mutation” refers to mutation of a humanchromosomal gene, the nucleotide sequence of which differs from awild-type nucleotide sequence (a nucleotide sequence of a normal gene).Further, although particular nucleotide sequence sites different fromperson to person are generally regarded as “genetic polymorphism,” inthe present invention these are classified as “genetic mutation.”“Genetic mutation” is identified by analyzing various aspects such asthe incidence rate of gene mutation, expression levels of its mRNA andprotein, or its protein function. It is generally regarded that, out ofseveral hundreds of nucleotides, on average one such “genetic mutation”occurs; and such a mutation can be identified through direct or indirectanalysis of the gene. Further, through analysis of the involved familytrees, whether such a genetic mutation is derived from the paternalchromosome allele or the maternal chromosome allele can also beidentified.

[0044] In the case of changes in a genetic mutation site, substitutionof a nucleotide base takes place at the corresponding site of either thepaternal allele and/or the maternal allele. When the nucleotide bases ofboth alleles are substituted and differ from the wild-type, it is calleda “homozygote,” and when the nucleotide base of only one allele issubstituted and differs from the wild-type, it is called a“heterozygote.”

[0045] As will be described later, the present inventors have so farfound the following genetic mutation sites correlated with risk factorsfor diabetic onset: (1) the site encoding arginine at amino acid residue140 of the CD38 protein encoded by the CD38 gene (for example, themutation is determined by the sensitivity of the exon 3 region to arestriction enzyme TspRI.); (2) the site encoding serine at amino acidresidue 264 (for example, the mutation is determined by the sensitivityof the exon 7 region to a restriction enzyme TaqI.); and (3) the siteencoding guanine at nucleotide position −28 in intron 7 (for example,the mutation is determined by the sensitivity of a region containing anintron 7 and exon 8 region to a restriction enzyme Tru9I.).

[0046] Concerning the detection methods of abnormal mutation sites,generally known methods can be used; for example, the RFLP method usingSouthern blotting, the PCR-RFLP method, the HET (heteroduplex analysis)method, the DGGE (denaturing gradient gel electrophoresis) method, theDS (direct sequencing) method, the CCM (chemical cleavage mismatch)method, the CDI (carbodiimide modification) method, the PCR-SSCP(single-stranded conformation polymorphism) method using the PCR method,which in the present specification is hereafter referred to as the SSCPmethod, and the PCR/GC-clamp method (for details, see, for example,Bio-Manual series I: “Basic Techniques for Gene Engineering,” TadashiYamamoto, ed., Yodosha, (1993); in particular, concerning thePCR/GC-clamp method, for example, see Myers, R. M., Shefield, V., andCox, D. R. (1988) in Genomic Analysis: A Practical Approach. K. Davies,ed. IRL Press Limited, Oxford, pp. 95-139). Among the above detectionmethods, the choice of the PCR/GC-clamp method is preferred because ofits easy handling and determination accuracy of genetic abnormalities.

[0047] PCR/GC-clamp method is a modified method of the DGGE method(which detects DNA nucleotide substitution based on a migrationdifference between a double-stranded DNA fragment with a substitutedbase(s) and that without a substituted base, since they migratedifferently due to their different migration speeds varying respectivelydepending on concentrations of a DNA-denaturation agent; in this case,on a linear concentration gradient of DNA-denaturation agent containedin a polyacrylamide gel). However, the DGGE method has a drawback inthat “in the case of the presence of substituted multiple bases in thetesting DNA fragments, the base substitution in a domain to be meltedlast in a polyacrylamide gel cannot be detected.” In contrast,PCR/GC-clamp method has overcome this problem by ligating a high GC-richregion (GC-clamp) to the testing DNA fragment (see Shefield, V. C. etal., (1989) Proc. Natl. Acad. Sci. USA 86: 232-236).

[0048] Thus, the operation of the PCR/GC-clamp method is basically thesame as that of the DGGE method (as is the case with the PCR/GC-clampmethod, see Myers, R. M., Shefield, V., and Cox, D. R. (1988) in GenomicAnalysis: A Practical Approach. K. Davies, ed. IRL Press Limited,Oxford, pp. 95-139), except that there is an additional step; i.e.ligating the GC-clamp to the testing DNA sample, required for detectionof base substitution.

[0049] The DNA sources needed in order to detect changes at particularsites carrying a possible genetic abnormality of the CD38 gene in themethod of the present invention are not particularly limited, so long asthey are derived from somatic cells of subjects. For example, bloodsamples such as peripheral blood or leukocytes can be successfully usedfor the method of the present invention.

[0050] Genomic DNA is extracted from testing cells of individualsubjects using a conventional method(s). By use of the extracted genomicDNA, changes at particular sites carrying a possible genetic abnormality(to be precise, base substitution at particular sites with a possiblegenetic abnormality) are detected.

[0051] Then, when such changes are detected, in consideration of thechanges in combination with clinical symptoms of the subjects, the riskfactors for diabetic onset of the subject can be determined.

[0052] That is, in the case of the subject already experiencing diabeticonset, the major cause for diabetic onset can be identified. Further, ifthe symptoms indicate non-insulin dependent diabetes, whether or not thediabetes will proceed to insulin-dependent diabetes can be predicted. Asa result, treatments suitable for the symptoms or preventive measurescan be provided, or the optimum treatment method can be developed.

[0053] Alternatively, in the case of the subject who has not yetexperienced diabetic onset or who has been determined to belong to therisk group on the basis of results of a different examination or familymedical history, proper preventive measures against the onset ofdiabetes, such as practical guidance for diet and exercise, can beprovided by regarding changes at particular sites carrying a possiblegenetic abnormality as a genetic diabetes constitution of the subject.In this way, diabetic onset in the subject may be prevented.

EXAMPLES

[0054] The present invention will next be described in detail by way ofexamples, which should not be construed as limiting the presentinvention.

[0055] Analysis of the CD38 Gene

[0056] Subjects

[0057] Mutation analysis of the CD38 gene was carried out with the DGGEmethod using 240 subjects who had been diagnosed as diabetes based onthe diagnosis criteria proposed by the Japan Diabetes Society.

[0058] Extraction of Genomic DNA

[0059] After collecting peripheral blood from the diabetic patientsusing blood-collecting vacuum tubes containing an anti-coagulation agentEDTA 3K, genomic DNA from healthy persons and the diabetic subjects wererespectively extracted by use of a QIAamp Blood Kit (QIAGEN).

[0060] Set-up of PCR Primers and Electrophoresis Conditions for the DGGEAnalysis

[0061] DNA sequence of the CD38 gene (Sequence No. 1) encoding the humanCD38 protein, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase, wasobtained from DDBJ/EMBL/GenBank databases (ACCESSION D84278-84284; Nata,K., et al.).

[0062] Primers for amplifying each exon of the CD38 gene using the PCRmethod were prepared by use of computer software GENETYX-MAC (SoftwareDeveloping Co. Ltd.). Characteristics (domain positions, meltingtemperatures, etc.) of melting domains of a DNA fragment amplified withthe PCR primers were predicted by use of computer software Mac Melt(Nippon Bio-Rad Laboratories K.K.). To which primers a GC-clamp shouldbe ligated was predicted from the melting characteristics obtained bycomputation, and further an optimal concentration range of adenaturation agent suitable for analysis of the resultant PCR DNAfragment was determined.

[0063] The nucleotide sequences of PCR primers used for amplification ofrespective exons of the CD38 gene are shown below: Exon 1 5′-CGC CCG CCGCGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GAT CTT CGC CCA GCC AAC CCC G-3′(Forward: Sequence No. 3) 5′-ACC GGT GCG CCT TAG TCG CCA-3′ (Reverse:Sequence No. 4) Exon 2 5′-TAG ACT GCA TGT TAG ACG AGA-3′ (Forward:Sequence No. 5) 5′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCCGTT TGG (Reverse: Sequence No. 6) ACC TAT GAA TTG TTA CC-3′ Exon 35′-GAC ATG CTA AAT TGA TCT CAG-3′ (Forward: Sequence No. 7) 5′-CGC CCGCCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GCA GCA (Reverse: SequenceNo. 8) GAA GTC ACT CTG TTC-3′ Exon 4 5′-CCA TTC TCC AGC CTC CGT CTT-3′(Forward: Sequence No. 9) 5′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCCCCG CCC GCA AGC (Reverse: Sequence No. 10) ACT GAC TGA GTA ACG TC-3′Exon 5 5′-AAA CTG CTG GAG GAT GGT GAT T-3′ (Forward: Sequence No. 11)5′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GTT CAC (Reverse:Sequence No. 12) TGT GAT ATT TGC AAC AGG-3′ Exon 6 5′-GGT TGA TGT TTGGGG TTC TTT GT-3′ (Forward: Sequence No. 13) 5′-CGC CCG CCG CGC CCC GCGCCC GTC CCG CCG CCC CCG CCC GTG TGG (Reverse: Sequence No. 14) ATT CTTTTG TGG ACT GAT T-3′ Exon 7 5′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCGCCC CCG CCC GTT GTC (Forward: Sequence No. 15) CAG GGC GTG CTA CAA A-3′5′-AGA TTC ACA CAG CCC TCC AAG-3′ (Reverse: Sequence No.16) Exon 85′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GTT AGC (Forward:Sequence No. 17) GAA TTG GAC GAC AGA TG-3′ 5′-TCT GGC ATT GAC CTT ATTGTG G-3′ (Reverse: Sequence No. 18) Exon 3 5′-CTC CGC CAC TCT CCT GCACAC A-3′ (Forward: Sequence No. 19) 5′-GGG CCT CCA CCA GAA GTC AC-3′(Reverse: Sequence No. 20) Exon 7 5′-TTG TCC AGG GCG TGC TAC AAA-3′(Forward: Sequence No. 21)

[0064] PCR Amplification

[0065] By use of 0.5 μg/μL extracted genomic DNA, PCR amplification ofeach exon was carried out in a 50 μL PCR reaction mixture [10 mMTris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂] which contained 0.8μM synthetic oligonucleotide primer for each exon, 200 μM nucleotidetriphosphate (dNTP) and 3% formamide and to which 0.5 units of Taq DNApolymerase (Perkin Elmer) had been added under the followingamplification conditions: 40 cycles of a series of reactions at 94° C.for 30 sec, at 54-68° C. for 30 sec, and at 72° C. for 1 min, followedby the last 10 min reaction at 72° C., to thereby obtain PCR products.Confirmation of the PCR reaction was carried out by performing 3%agarose gel electrophoresis using each 5 μL PCR product followed bystaining with 0.5 μg/mL ethidium bromide.

[0066] Note that each exon of the CD38 gene corresponds to the followingnucleotide sequence positions: exon 1, nucleotide positions 1-233 ofSequence No. 1; exon 2, 234-363 of Sequence No. 1; exon 3, 364-498 ofSequence No. 1; exon 4, 499-585 of Sequence No. 1; exon 5, 586-659 ofSequence No. 1; exon 6, 660-752 of Sequence No. 1; exon 7, 753-839 ofSequence No. 1; and exon 8, 840-890 of Sequence No. 1.

[0067] Screening of Genetic Mutations by the DGGE Method

[0068] DGGE method was carried out by following the protocol designed byMyers, et al. The denaturing gradient polyacrylamide gel was prepared byuse of glass plates (177×220 mm)(Takara Shuzo, Co. Ltd.). The optimalconcentration of a denaturing agent was chosen for each exon, and eachconcentration gradient was designed to be within a range of 30%. Eachpolyacrylamide gel containing an optimal concentration gradient of adenaturing agent was prepared by a gradient maker, mixing 9% acrylamide(acrylamide:bis-acrylamide =37.5:1) in TAE solution (40 mM Tris, 20 mMsodium acetate, and 2 mM EDTA (pH 7.4)) containing 0% denaturing agentand 9% acrylamide in TAE solution containing 80% denaturing agent (5.19M urea and 30% deionized formamide).

[0069] Screening genetic mutations of the PCR products by the DGGEmethod was carried out as follows: after aliquoting 15 μL of theabove-mentioned PCR product from each exon to a 500-μL tube, the samplewas dried in a vacuum dryer; to each sample tube, 10 μL loading buffer[20% Ficoll, 1 mM EDTA, and 0.5% bromophenol blue in 10 mM Tris-HClbuffer (pH 7.8)] was added; after dissolving, 5 μL sample was eachapplied to the gel. Electrophoresis was carried out under conditions of150 V×16 h in a DDGE electrophoretic chamber (Takara Shuzo, Co. Ltd.)containing 1×TAE solution (16 L) kept at 60° C. After electrophoresis,the gel was subjected to staining with 0.5 μg/mL ethidium bromide, andthen DNA was visualized under UV light, followed by pattern analysis.

[0070] The results obtained by screening mutation in each exon andintron region of CD38 genes using the DGGE method showed that there wereband patterns differing from the wild type found 1 at exon 2 (FIG. 1), 2at exon 3 (FIG. 2), 2 at exon 4 (FIG. 3), 1 at exon 7 (FIG. 4), and 1 atexon 8 (FIG. 5). These were confirmed to be abnormal band patterns.Concerning the gene abnormality, the obtained abnormal band patternsdemonstrated by the DGGE method suggest that the corresponding PCRproducts contain nucleotide sequences different from the wild type.Determination of nucleotide sequences thereof would have to be carriedout by another method.

[0071] Determination of Nucleotide Sequences by Direct Sequencing

[0072] The PCR products with abnormal patterns shown by the DGGE methodwere subjected to DNA sequencing with an automated sequencer using adirect sequencing method. The PCR products showing the abnormal patternswere fluorescence-labeled by use of BigDye Terminator cycle sequencingFs Ready Reaction Kit (Perkin Elmer). Then, the samples were subjectedto nucleotide sequencing by use of ABI PRISM 377 DNA sequencer (AppliedBiosystems).

[0073] The direct sequencing results obtained on an automated sequencerwere subjected to analysis, showing that the individual exhibiting theabnormal band pattern for exon 2 carried thymine (T) besides thewild-type cytosine (C) at nucleotide position 348. Thus, the basesubstitution of C with T was identified at nucleotide position 348 (FIG.6). Consequently, due to the single base substitution from C to T, thecorresponding codon at 116 amino acid residue was changed from ACC toACT. However, the encoded amino acid residue was the same threonine(Thr), resulting in no amino acid substitution due to the single basesubstitution. Thus, the base substitution from C to T at nucleotideposition 348 turned out to be not accompanied with amino acidsubstitution, a so-called silent mutation, and the individual exhibitingthe abnormal pattern carried the heterozygote with C348T mutation.

[0074] One of individuals exhibiting an abnormal pattern for exon 3showed T at nucleotide position 418 in addition to the wild-type C. Dueto the one-base substitution from C to T, the corresponding codon atamino acid residue 140 was converted from CGG to TGG, resulting in thechange at amino acid residue 140 from arginine (Arg) to tryptophan(Trp), which is shown in FIG. 7. Thus, the individual with the abnormalband pattern for exon 3 carried one amino acid substitution Arg140Trp atamino acid residue 140, a so-called missense mutation, and carried theheterozygote with Arg140Trp mutation.

[0075] Sequencing of DNA from the other individual exhibiting anabnormal pattern for exon 3 showed only T base at nucleotide position418, and the individual turned out to carry the homozygote withArg140Trp mutation (FIG. 8).

[0076] One of individuals with an abnormal pattern for exon 4 showed Cat nucleotide position 504 in addition to the wild-type A. Due toone-base substitution from A to C, the corresponding codon at amino acidresidue 168 was changed from ATA to ATC, as shown in FIG. 9. However,the corresponding amino acid at amino acid residue 168 remained the sameas isoleucine (Ile). Thus, the one-base substitution from A to C atnucleotide position 504 belonged to the silent mutation, and theindividual with the abnormal pattern for exon 4 carried the heterozygotewith A504C mutation.

[0077] Another individual with an abnormal pattern for exon 4 showedonly C at nucleotide position 504, thus carrying the homozygote withA504C mutation (FIG. 10).

[0078] An individual exhibiting an abnormal pattern for exon 7 showed Tin addition to the wild-type C at nucleotide position 791. Due to theone base substitution from C to T, the corresponding codon at amino acidresidue 264 was changed from TCG to TTG, resulting in one amino acidsubstitution from serine (Ser) to leucine (Leu) (FIG. 11). Thus, theindividual exhibiting the abnormal pattern for exon 7 carried theheterozygote with the amino acid substitution of Ser264Leu at amino acidresidue 264 due to one-base substitution at nucleotide position 791 fromC to T.

[0079] An individual exhibiting an abnormal pattern for exon 8 showed Ain addition to wild-type G within intron 7 upstream of exon 8 of the CDgene, at −28 base position from the splicing acceptor site of exon 8(Sequence No. 22: base position 39 of the intron 7 sequence) (FIG. 12).Thus, the abnormal pattern detected with exon 8 was due to one-basesubstitution from G to A at base position −28 upstream of theabove-mentioned acceptor site, resulting in the heterozygote with G/Amutation at −28 base position within intron 7.

[0080] As described above, concerning genetic mutations of the CD38 genedetected in the diabetes group, there were two missense mutations; i.e.,Arg140Trp at exon 3 and Ser264Leu at exon 7. In both cases, thecorresponding amino acid residues were changed, thus likely causingfunctional abnormalities in the CD38 protein. Indeed, the CD38 proteincarrying Arg140Trp mutation showed lower activity of both ADP-ribosylcyclase and cyclic ADP-ribose (cADPR) hydrolase (Diabetologia 41:1024-1028, 1998).

[0081] In contrast, in the case of Ser264Leu mutation, it is known thata region consisting of several amino acid residues including the aminoacid residue 264 plays an important role in binding to the substrateNAD⁺ upon expression of enzyme activity of the CD38 protein. Thus, theCD38 protein carrying an amino acid substitution at any amino acidresidue within the region including the above amino acid residue 264 isexpected to have lower enzymatic activity or lose the activity.

[0082] The −28 G/A mutation at intron 7 appears to be present within aconsensus sequence of the so-called “branched site,” that is involved information of the lariat structure upon mRNA splicing [Gene (the lastvolume), Tokyo Kagaku Dojin]. Thus, the −28 G/A mutation at intron 7likely affects generation of CD38 mRNA, and thereby could result insynthesis of a CD38 abnormal protein or abnormal mRNA of the CD38 gene.

[0083] Analysis of Incidence Rates of CD38 Gene Mutations

[0084] The present inventors have carried out comparative studies ofmutation incidence rates between the diabetic and healthy groups usingthe above-described mutations of the CD38 gene; i.e., C348T mutation atexon 2, Arg140Trp at exon 3, A504C at exon 4, Ser264Leu at exon 7, andthe −28G/A mutation at intron 7. Note that in the case of threemutations, Arg140Trp at exon 3, Ser264Leu at exon 7, and the −28G/Amutation at intron 7, sequences recognized by restriction enzymes werechanged due to one-base substitution. Thus, to detect the mutations, thePCR-RFLP method was used. In the case of C348T mutation at exon 2 andA504C at exon 4, the PCR-DGGE method was employed, as described above.

[0085] Gene Mutation Analysis by the PCR-RFLP Method

[0086] Detection Method for Exon 3 Arg40Trp

[0087] Using 0.5 μL extracted genomic DNA and oligonucleotide primers ofSequence Nos. 19 and 20, PCR reactions were carried out to obtain a PCRproduct of 381-bp fragment, as described above. After digestion of 10 μLof the resultant PCR product with 2.5 units TspRI restriction enzyme(New England BioLabs) at 65° C. overnight, the digested sample wassubjected to 3% agarose gel electrophoresis to detect Arg140Trp at exon3. The wild-type 381-bp DNA fragment was digested into 2 fragments of311 and 70 bp, whereas the DNA fragment carrying Arg140Trp mutation wassplit into 3 fragments of 228, 83, and 70 bp (FIG. 13). In this way, thepresence of the mutation was detected.

[0088] Detection Method for Exon 7 Ser264Leu

[0089] By use of 0.5 μL extracted genomic DNA, oligonucleotide primersof Sequence Nos. 17 and 21, and PCR solution containing the finalconcentration of 3% formamide, PCR reactions were carried out to obtaina PCR product of 274-bp fragment, as described above. After digestion of10 μL of the resultant PCR product with 5 units TaqI restriction enzyme(New England BioLabs) at 65° C. overnight, the digested sample wassubjected to 3% agarose gel electrophoresis to detect the exon 7Ser264Leu mutation. The wild-type 274-bp DNA fragment was digested into2 fragments of 149 and 125 bp, whereas the DNA fragment carryingSer264Leu mutation was not digested (FIG. 14). In this way, the presenceof the mutation was detected.

[0090] Detection Method for Intron 7 −28 G/A Mutation

[0091] Using 0.5 μL extracted genomic DNA, oligonucleotide primers ofSequence Nos. 17 and 18, and PCR solution containing the finalconcentration of 3% formamide, PCR reactions were carried out to obtaina PCR product of 297-bp fragment, as described above. After digestion of10 μL of the resultant PCR product with 2 units Tru9I restriction enzyme(Promega) at 65° C. overnight, the digested sample was subjected to 3%agarose gel electrophoresis to detect the intron 7 −28 G/A mutation. Thewild-type 297-bp DNA fragment was not digested at all, whereas the DNAfragment carrying the intron 7 −28 G/A mutation was digested into 2fragments of 220 and 77 bp (FIG. 15). In this way, the presence of themutation was detected.

[0092] Incidence Rate of Each Genetic Mutation

[0093] The present inventors have analyzed a total of 757 diabeticpatients, including 240 cases analyzed genetically by the DGGE method.

[0094] The exon 2 C348T mutation was found in 2 out of 240 cases, andits incidence rate was 0.8%. In the case of the exon 3 Arg140Trpmutation, the heterozygote was observed in 27 out of 757 cases, and thehomozygote was found in 1 out of 757 cases. Thus, the total incidencerate was 3.7%. In the case of the exon 4 A504C mutation, theheterozygote was found in 58 out of 240 cases, and the homozygote wasfound in 4 out of 240 cases, showing the total incidence rate of 25.8%.For the exon 7 Ser264Leu mutation, the heterozygote was found in 9 outof 757 cases, showing the incidence rate of 1.2%. For the intron 7 −28G/A mutation, the heterozygote was found in 9 out of 757 cases, showingthe incidence rate of 1.2%.

[0095] In contrast, when 205 non-diabetic subjects were analyzed, 3cases showed the heterozygote for the exon 3 Arg140Trp mutation (3/205cases), 2 cases showed the heterozygote for the intron 7 −28 G/Amutation (2/205 cases), and 0 cases showed the exon 7 Ser264Leu mutation(0/205 cases).

[0096] In 2 cases showing the heterozygote for the exon 2 C348Tmutation, one case showed the heterozygote for the A504C mutation,whereas the other showed no such A504C mutation but rather the wild-typebase sequence. Moreover, out of 58 cases showing the heterozygote forthe A504C mutation, 57 cases did not carry any other mutation. Fromthese results, it is possible that the C348T mutation and the A504Cmutation respectively reside on independent alleles.

[0097] As shown above, the present inventors have analyzed mutations ofthe gene encoding the CD38 protein involving insulin secretion atinsulin-producing cells. Then, after obtaining the results, the presentinventors have further carried out comparative studies between thediabetic and non-diabetic groups for incidence rates of geneticmutations of the gene. The obtained results show that in the case of themutations of exon 3 Arg140Trp, exon 7 Ser264Leu, and intron 7 −28 G/A,all of which at least will probably cause loss of expression andfunction of the CD38 protein, the total incidence rate of thosemutations was 6.1% (46/757 cases), demonstrating that the rate wassignificantly high as compared with 2.4% of the non-diabetic group(5/205 cases). It has been reported that the total mutation incidencerate of the mitochondrial gene in the diabetic group was about 2%, whosemutations are regarded as diabetic causing mutations within a singlegene. In contrast, the mutation incidence rate of the CD38 gene encodingthe CD38 protein was 6.1% for the diabetic group, indicating that themutation incidence rate of the CD38 gene was extremely high in thediabetic group as compared with that of the mitochondrial gene. Further,when this was compared with other incidence rates reported so far, theincidence rate of 6.1% was likewise outstandingly high. It is well knownthat particular mutations could accumulate or be distributed unevenlyfrom country to country or from region to region. Carrying out suchstudies and analysis of the diabetic subjects on a large scale may leadto finding much higher incidence rates of those 3-type genetic mutationsor to finding of some other mutations of the CD38 gene other than the3-type genetic mutations. Furthermore, use of the sum of the totalabnormalities of the CD38 gene could identify a much higher number ofdiabetic patients than separate use of each mutation. Thus, the above3-type genetic mutations or possible new mutations should not be usedseparately, but should be handled as mutations of the CD38 gene as thewhole, which inevitably would greatly enhance usefulness of thosemarkers for detecting risk factors for diabetic onset.

[0098] [Effects of the Invention]

[0099] Accorindg to the present invention, the means for detecting riskfactors for diabetic onset using a gene is provided.

1 22 1 1408 DNA Hominidae CDS (1)..(900) 1 aaa cagaagggga ggtgcagtttcagaacccag ccagcctctc 43 tcttgctgcc tagcctcctg ccggcctcat cttcgcccagccaaccccgc ctggagccct 103 atg gcc aac tgc gag ttc agc ccg gtg tcc ggggac aaa ccc tgc tgc 151 Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly AspLys Pro Cys Cys 1 5 10 15 cgg ctc tct agg aga gcc caa ctc tgt ctt ggcgtc agt atc ctg gtc 199 Arg Leu Ser Arg Arg Ala Gln Leu Cys Leu Gly ValSer Ile Leu Val 20 25 30 ctg atc ctc gtc gtg gtg ctc gcg gtg gtc gtc ccgagg tgg cgc cag 247 Leu Ile Leu Val Val Val Leu Ala Val Val Val Pro ArgTrp Arg Gln 35 40 45 cag tgg agc ggt ccg ggc acc acc aag cgc ttt ccc gagacc gtc ctg 295 Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu ThrVal Leu 50 55 60 gcg cga tgc gtc aag tac act gaa att cat cct gag atg agacat gta 343 Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg HisVal 65 70 75 80 gac tgc caa agt gta tgg gat gct ttc aag ggt gca ttt atttca aaa 391 Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile SerLys 85 90 95 cat cct tgc aac att act gaa gaa gac tat cag cca cta atg aagttg 439 His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys Leu100 105 110 gga act cag acc gta cct tgc aac aag att ctt ctt tgg agc agaata 487 Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp Ser Arg Ile115 120 125 aaa gat ctg gcc cat cag ttc aca cag gtc cag cgg gac atg ttcacc 535 Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg Asp Met Phe Thr130 135 140 ctg gag gac acg ctg cta ggc tac ctt gct gat gac ctc aca tggtgt 583 Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp Leu Thr Trp Cys145 150 155 160 ggt gaa ttc aac act tcc aaa ata aac tat caa tct tgc ccagac tgg 631 Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser Cys Pro AspTrp 165 170 175 aga aag gac tgc agc aac aac cct gtt tca gta ttc tgg aaaacg gtt 679 Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys ThrVal 180 185 190 tcc cgc agg ttt gca gaa gct gcc tgt gat gtg gtc cat gtgatg ctc 727 Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp Val Val His Val MetLeu 195 200 205 aat gga tcc cgc agt aaa atc ttt gac aaa aac agc act tttggg agt 775 Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe GlySer 210 215 220 gtg gaa gtc cat aat ttg caa cca gag aag gtt cag aca ctagag gcc 823 Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Thr Leu GluAla 225 230 235 240 tgg gtg ata cat ggt gga aga gaa gat tcc aga gac ttatgc cag gat 871 Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp Leu CysGln Asp 245 250 255 ccc acc ata aaa gag ctg gaa tcg att ata agc aaa aggaat att caa 919 Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys Arg AsnIle Gln 260 265 270 ttt tcc tgc aag aat atc tac aga cct gac aag ttt cttcag tgt gtg 967 Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe Leu GlnCys Val 275 280 285 aaa aat cct gag gat tca tct tgc aca tct gag atctgagccagtc 1013 Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser Glu Ile 290 295300 gctgtggttg ttttagctcc ttgactcctt gtggtttatg tcatcataca tgactcagca1073 tacctgctgg tgcagagctg aagattttgg agggtcctcc acaataaggt caatgccaga1133 gacggaagcc tttttcccca aagtcttaaa ataacttata tcatcagcat acctttattg1193 tgatctatca atagtcaaga aaaattattg tataagatta gaatgaaaat tgtatgttaa1253 gttacttcac tttaattctc atgtgatcct tttatgttat ttatatattg gtaacatcct1313 ttctattgaa aaatcaccac accaaacctc tcttattaga acaggcaagt gaagaaaagt1373 gaatgctcaa gtttttcaga aagcattaca tttcc 1408 2 300 PRT Hominidae 2Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys 1 5 1015 Arg Leu Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu Val 20 2530 Leu Ile Leu Val Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln 35 4045 Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu 50 5560 Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val 65 7075 80 Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser Lys 8590 95 His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys Leu100 105 110 Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp Ser ArgIle 115 120 125 Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg Asp MetPhe Thr 130 135 140 Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp LeuThr Trp Cys 145 150 155 160 Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr GlnSer Cys Pro Asp Trp 165 170 175 Arg Lys Asp Cys Ser Asn Asn Pro Val SerVal Phe Trp Lys Thr Val 180 185 190 Ser Arg Arg Phe Ala Glu Ala Ala CysAsp Val Val His Val Met Leu 195 200 205 Asn Gly Ser Arg Ser Lys Ile PheAsp Lys Asn Ser Thr Phe Gly Ser 210 215 220 Val Glu Val His Asn Leu GlnPro Glu Lys Val Gln Thr Leu Glu Ala 225 230 235 240 Trp Val Ile His GlyGly Arg Glu Asp Ser Arg Asp Leu Cys Gln Asp 245 250 255 Pro Thr Ile LysGlu Leu Glu Ser Ile Ile Ser Lys Arg Asn Ile Gln 260 265 270 Phe Ser CysLys Asn Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys Val 275 280 285 Lys AsnPro Glu Asp Ser Ser Cys Thr Ser Glu Ile 290 295 300 3 61 DNA Hominidae 3cgcccgccgc gccccgcgcc cgtcccgccg cccccgcccg atcttcgccc agccaacccc 60 g61 4 21 DNA Hominidae 4 accggtgcgc cttagtcgcc a 21 5 21 DNA Hominidae 5tagactgcat gttagacgag a 21 6 62 DNA Hominidae 6 cgcccgccgc gccccgcgcccgtcccgccg cccccgcccg tttggaccta tgaattgtta 60 cc 62 7 21 DNA Hominidae7 gacatgctaa attgatctca g 21 8 60 DNA Hominidae 8 cgcccgccgc gccccgcgcccgtcccgccg cccccgcccg cagcagaagt cactctgttc 60 9 21 DNA Hominidae 9ccattctcca gcctccgtct t 21 10 62 DNA Hominidae 10 cgcccgccgc gccccgcgcccgtcccgccg cccccgcccg caagcactga ctgagtaacg 60 tc 62 11 22 DNA Hominidae11 aaactgctgg aggatggtga tt 22 12 63 DNA Hominidae 12 cgcccgccgcgccccgcgcc cgtcccgccg cccccgcccg ttcactgtga tatttgcaac 60 agg 63 13 23DNA Hominidae 13 ggttgatgtt tggggttctt tgt 23 14 64 DNA Hominidae 14cgcccgccgc gccccgcgcc cgtcccgccg cccccgcccg tgtggattct tttgtggact 60gatt 64 15 61 DNA Hominidae 15 cgcccgccgc gccccgcgcc cgtcccgccgcccccgcccg ttgtccaggg cgtgctacaa 60 a 61 16 21 DNA Hominidae 16agattcacac agccctccaa g 21 17 62 DNA Hominidae 17 cgcccgccgc gccccgcgcccgtcccgccg cccccgcccg ttagcgaatt ggacgacaga 60 tg 62 18 22 DNA Hominidae18 tctggcattg accttattgt gg 22 19 22 DNA Hominidae 19 ctccgccactctcctgcaca ca 22 20 20 DNA Hominidae 20 gggcctccag cagaagtcac 20 21 21DNA Hominidae 21 ttgtccaggg cgtgctacaa a 21 22 66 DNA Hominidae 22ttagcgaatt ggacgacaga tgtatcctac ggtctcttga tttccttttt tgctttcttg 60tcatag 66

What is claimed is:
 1. A method of detecting a risk factor for diabeticonset in an individual through detection of genetic abnormality of geneCD38.
 2. A method of detecting a risk factor for diabetic onsetaccording to claim 1, wherein sites of abnormality of the gene CD38include the site encoding arginine at amino acid residue 140 of CD38protein encoded by the CD38 gene; the site encoding serine at amino acidresidue 264; and guanine at nucleotide position −28 in intron 7.